Expression of freezing and fear-potentiated startle during sustained fear in mice

Genes, Brain and Behavior (2015) 14: 281 291 doi: 10.1111/gbb.12211 Techniques Expression of freezing and fear-potentiated startle during sustained fear in mice T. Daldrup,1, J. Remmes,1, J. Lesting, S. Gaburro, M. Fendt,P.Meuth,V.Kloke, H.-C. Pape,1, and T. Seidenbecher,1, Institute of Physiology I, Westfälische Wilhelms-University Münster, Münster, Institute for Pharmacology and Toxicology & Center for Behavioral Brain Sciences, Otto-von-Guericke-University, Magdeburg, Department of Neurology and Institute of Neuropathophysiology, Westfälische Wilhelms-University Münster, and Department of Behavioural Biology, Westfälische Wilhelms-University Münster, Münster, Germany 1 These authors contributed equally to this work. *Corresponding authors: T. Seidenbecher or H.-C. Pape, Institut für Physiologie I, Westfälische Wilhelms-Universität Münster, Robert-Koch-Strasse, 27a, D-48149 Münster, Germany. E-mail: seidenbe@uni-muenster.de or papechris@ukmuenster.de Fear-potentiated acoustic startle paradigms have been used to investigate phasic and sustained components of conditioned fear in rats and humans. This study describes a novel training protocol to assess phasic and sustained fear in freely behaving C57BL/6J mice, using freezing and/or fear-potentiated startle as measures of fear, thereby, if needed, allowing in vivo application of various techniques, such as optogenetics, electrophysiology and pharmacological intervention, in freely behaving animals. An auditory Pavlovian fear conditioning paradigm, with pseudo-randomized conditioned unconditioned stimulus presentations at various durations, is combined with repetitive brief auditory white noise burst presentations during fear memory retrieval 24 h after fear conditioning. Major findings are that (1) a motion sensitive platform built on mechano-electrical transducers enables measurement of startle responses in freely behaving mice, (2) absence or presence of startle stimuli during retrieval as well as unpredictability of a given threat determine phasic and sustained fear response profiles and (3) both freezing and startle responses indicate phasic and sustained components of behavioral fear, with sustained freezing reflecting unpredictability of conditioned stimulus (CS)/unconditioned stimulus (US) pairings. This paradigm and available genetically modified mouse lines will pave the way for investigation of the molecular and neural mechanisms relating to the transition from phasic to sustained fear. Keywords: Anxiety, behavior, conditioning, fear, freely moving, learning, mouse, predictability, retrieval, unpredictability Received 16 January 2015, revised 20 February 2015, accepted for publication 9 March 2015 Anxiety disorders are serious medical illnesses, which have the highest life time prevalence (19 29%) of psychiatric diseases with high economical costs for health systems (Kaufman & Charney 2000; Kessler et al. 2005; Wittchen et al. 2011). Consequently, animal models of anxiety have been extensively investigated to understand neuronal mechanisms of psychiatric disorders in humans, including posttraumatic stress disorders (PTSD), phobias and panic attacks. Much of our understanding of these fear and anxiety related mechanisms is based on animal studies in rodents using Pavlovian paradigms (LeDoux 1993; for review, see Pape & Pare 2010). In such a conditioned fear paradigm, a neutral stimulus (e.g. tone or light) is paired with an aversive stimulus (e.g. electric footshock) to evaluate short-term as well as long-term conditioned fear responses to the predictable conditioned threat. Thus, after Pavlovian fear conditioning, presentation of the conditioned stimulus (CS) alone has the ability to generate a variety of behavioral expressions, such as freezing and/or fear-potentiated startle responses, indicating states of fear and anxiety. Fear is a generally adaptive state of apprehension that develops rapidly and declines quickly once the threatening stimulus is absent; a physiological situation reflecting a phasic fear state, which can be measured after training to short and discrete cues that are predictably paired with aversive events (e.g. footshock) (Davis et al. 1989; Davis et al. 2010; de Jongh et al. 2003; Miles et al. 2011). In contrast to that, situations in patients suffering from anxiety disorders are characterized by a more long-lasting state of fear. It is, therefore, assumed that clinical symptoms of several anxiety disorders can be modeled more precisely with paradigms aiming on these long-lasting and sustained, rather than phasic fear states. To account for that, experimental studies have begun to focus on more long-lasting states of fear, that are elicited by less predictable threats using fear-potentiated startle as behavioral readout in animals and humans (Alvarez et al. 2011; for review, see Davis et al. 2010). These fear states can be measured after training to more diffuse (non-associating) stimuli, arranged so that the subject cannot predict the occurrence of the aversive event. Accordingly, Walker and Davis (2008) developed a paradigm in rats, in which short- or long-duration clicker stimuli were paired with footshocks in 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society 281

Daldrup et al. a pseudo-randomized fashion, and acoustic startle amplitude was measured in the absence of the clicker, or within seconds ( phasic fear ) or minutes ( sustained fear ) of its onset (reviewed in Davis et al. 2010). Results showed that unpredictability of the aversive event resulted in a sustained state of fear lasting throughout the CS with a slow decay after stimulus discontinuation. Subsequent studies have shown that phasic and sustained components of fear are pharmacologically dissociable (Miles et al. 2011) and involve distinctive regions of the extended amygdala (Walker et al. 2003, 2009). Importantly, these studies have been carried out in rats, and the amplitude of the acoustic startle reflex has been used as a measure of fear that has been found adequate to distinguish between phasic and sustained fear states (see Davis et al. 2010). Technicalities of startle measurement allowed for only minimal movement of the animals, and phasic and sustained fear states remain to be distinguished in freely moving animals. In addition, when it comes to mice, startle responses and fear-potentiated alterations of startle reflexes are notoriously difficult to study (Crawley & Paylor 1997). This is somewhat unfortunate, as numerous genetically modified mouse lines would provide a most promising potential for identification of the genetic, molecular and neuronal mechanisms of phasic vs. sustained fear states and for the development of specific intervention strategies. Therefore, the major aims of the present study have been twofold: (1) to design and validate an apparatus allowing recording of startle responses in freely moving (non-restrained) mice and (ii) to develop a fear training paradigm involving non-anticipated CS allowing to distinguish phasic and sustained fear states in mice. Animals, material and methods All experiments were performed in accordance to the European Communities Council directive (86/609/EEC), with the regulations of German law and as approved by the local animal care committee of LANUV NRW (AZ 84 02.04.2012.A206). Animals were kept in a 12-h light/dark cycle provided with food (Altromin 1324, Altromin GmbH, Lage, Germany) and water ad libitum. The experiments were conducted with 9 12 weeks old male C57BL/6J mice (n = 70, M&B Taconic, Berlin, Germany). All animals were housed in transparent standard Macrolon cages type III with sawdust as bedding material (Allspan, Höveler GmbH & Co.KG, Langenfeld, Germany). After weaning at day 21 ± 1 of age, the mice were kept in groups of two to five same-sex littermates until the age of 71 ± 11 days, before they were separated from their littermates and single housed for the rest of the experiment. One week after separation, mice underwent behavioral experiments (pre-test, adaptation, fear conditioning, retrieval). Before an experimental session, mice were carried in their home cage from the central animal facility to separate rooms that contained the experimental equipment (room A/context A for pre-testing, adaptation and retrieval; room B/context B for fear conditioning). The sustained fear protocol consisted of pre-testing (stimulus-response curve), Figure 1: Training paradigm for phasic/sustained components of fear in mice. (a) Individual startle responsiveness is assessed through white noise burst presentations at different intensities. Arrows symbolize pseudo-randomized burst presentations at six different burst intensities, each presented five times. (b) Following adaptation to 36 startle-inducing bursts on day 1, (c) mice are exposed to two times four conditioned stimuli (CS) on the next day (day 2), which are presented at pseudo-randomized duration, paired with footshock after CS termination (inter-session interval 6 h). (d) During fear memory retrieval (day 3), white noise bursts (total of 36) are presented before, during and after presentation of the CS (each period 6 min). Black arrows indicate white noise bursts, horizontal bar indicates CS, open arrows indicate footshocks. adaptation (pre-conditioning), auditory fear conditioning and post-conditioning test (retrieval) with a 24-h delay between each session. A schematic drawing of the experimental paradigm is illustrated in Fig. 1 (experimental paradigm for control groups differed in training session as schematically shown in Fig. 5 and described below). 282 Genes, Brain and Behavior (2015) 14: 281 291

Phasic and sustained fear in mice Quantification of freezing and fear-potentiated startle in mice The experimental paradigm described here has been used to measure freezing (immobility except for respiratory movements) and fear-potentiated startle as indicators of behavioral fear. Freezing was evaluated off-line by an experienced experimenter (naive to experimental conditions) using video file recordings and scored in percent per 30 seconds time bins across experimental period. Startle stimuli, evoked by 50 ms white noise bursts, were generated by a TTL-triggered custom-made sound generator and delivered through a speaker located 30 cm above the test box. The startle amplitude in an individual was quantified using a motion-sensitive platform equipped with three piezo buzzer diaphragms (EPZ-20, 6.4 khz, 400 ohm, 35 000 pf; Bürklin, Germany) mounted at the bottom of a polyvinyl chloride (PVC) plate (length: 40, width: 25, height: 2 cm) to transduce the animal s motion (e.g. exploration and startle) into a voltage signal (fivefold amplified, band-pass filtered from 1 to 500 Hz, DPA-2F, Science Products GmbH, Hofheim, Germany). Startle amplitude was defined as maximum of the peak-to-trough voltage signal occurring within the first 100 ms after onset of each white noise burst presentation and digitized into arbitrary values by Spike2 software (version 7, Science Products GmbH, Hofheim, Germany) (see analog example Fig. 2a). A standard Macrolon cage type III was horizontally separated into two parts (see Fig. 2a,b). One part was placed on the motion-sensitive platform and was used as an open field-like arena (length: 33, width: 17.5, height: 3.5 cm). The second part (length: 33, width: 17.5, height: 10 cm) was positioned above the movable first part and served as arena walls. The integrated signal of the piezo elements produced a voltage output proportional to the arena movement (see analog example in Figs. 2, 3b,c). A schematic drawing and a photograph of the motion sensitive platform are shown in Fig. 2 illustrating the fixed part (A) and the sensitive movable part (B). Fear-potentiated startle was defined operationally as greater startle amplitude to the startle-provoking burst during presence vs. absence of a CS. Pre-test (stimulus-response curve) This test was performed to evaluate the stimulus threshold of startle-provoking bursts (day 0). Each single animal was presented with white noise bursts at different intensities (55 95 db in 10 db steps and 100 db in addition, 50 ms duration, 0 ms rise/fall time) to identify the mean maximum startle response. Each stimulus was delivered five times pseudo-randomly with an inter-stimulus interval (ISI) of 30 seconds (see Fig. 1a). The burst intensity which evoked 50% of the mean maximum startle response was defined as standard intensity upon experimental procedure (see Fig. 3a,b). Adaptation (pre-conditioning) The pre-conditioning test (day 1, Fig. 1b) was conducted to evaluate an individual baseline level of freezing and startle response of the animals before fear conditioning and testing. Mice were adapted to the test cage for 1 min (context A, motion sensitive platform as described) followed by Figure 2: Motion sensitive platform for measurement of startle responses. (a) Schematic drawing illustrating the construction of the motion sensitive platform. The apparatus is divided in a stationary (fixed) part (A; length: 33, width: 17.5, height: 10 cm) and a motion-sensitive movable part (B; length: 33, width: 17.5, height: 3.5 cm). Motions are detected by three piezo elements whose individual signals are integrated into a combined signal reflecting movement of the animal. Startle responses are measured as peak-to-trough amplitude of the combined signal (in arbitrary units). The original trace shows an example of a startle response to a 50 ms white noise burst. (b) Photograph illustrating the motion-sensitive platform during behavioral recording of a freely behaving mouse. 36 startle-eliciting white noise bursts (individual intensity pre-determined on day 0, 50 ms duration, inter-burst interval 30 seconds) (see analog example of unconditioned startle response during adaptation in Fig. 3c). The mean of all 36 startle amplitudes was used as reference value for z-scored fear-potentiated startle responses during fear retrieval. Fear conditioning On day 2, fear conditioning was performed in a standardized fear conditioning chamber (context B, Fear Conditioning System, TSE, Bad Homburg, Germany). All animals underwent Genes, Brain and Behavior (2015) 14: 281 291 283

Daldrup et al. Figure 3: Examples of behavioral responses (startle and freezing) during pre-test, adaptation and retrieval session. (a) Assessment of startle responses to white noise burst presentations at different intensities. Mean maximum startle amplitude (solid horizontal arrow) was taken to determine the standard stimulus intensity (vertical arrow) at a 50% threshold (dashed horizontal arrow). (b) Mean startle responses of all animals during pre-testing at different intensities. (c) Startle amplitudes to 36 white noise bursts during adaptation indicate less (and adapting) startle responses to stimulus presentations. (d) Startle amplitudes to 36 white noise bursts during retrieval session indicate increased startle responses to stimuli during presentation of the conditioned stimulus 24 h after fear conditioning. Note reduced movement activity in the recording trace upon CS presentation (gray horizontal bar), reflecting off-line scored freezing behavior (black horizontal bars on top). the following fear conditioning protocol: mice were given a 2-min acclimation to the fear conditioning apparatus followed by the presentation of four 10 khz tones as CS (75 db, pseudo-randomized stimulus presentation with variable duration of 29, 9, 19 and 14 seconds, ISI 30 seconds between CS termination and onset of the next CS). An unconditioned stimulus (US, scrambled footshock, 0.4 ma, duration 1 seconds) coincided with the termination of each CS (see schematic drawing in Fig. 4a). A second session was repeated 6 h later with CS duration in altered order (19, 14, 29 and 9 seconds) (see Fig. 1c). Testing (retrieval) Twenty-four hours after fear conditioning (day 3), single animals (main experimental group: CS + burst, n = 18) were transferred to the retrieval environment (context A), habituated over a period of 1 min before being exposed to overall 36 (calibrated) white noise bursts (50 ms duration, inter-burst interval 30 seconds), being divided into 12 bursts in absence, 12 bursts in presence and again 12 bursts in absence of the CS (duration of each part 6 min), followed by another minute after the last burst presentation (see analog example of a retrieval session with fear-conditioned startle responses in Fig. 3d). A 75 db, 10 khz sine wave stimulus was used as CS, delivered through a second speaker located 30 cm above the test box. The first startle stimulus occurred 20 seconds after CS onset. Startle amplitudes were z-scored to the mean amplitude of 36 white noise bursts, which were identified during adaptation. As control, in a separate group of C57BL/6J, retrieval sessions were performed as described above but without presenting white noise bursts (CS-only group; n = 12). Freezing behavior (% of time averaged in 30 seconds bins) and fear-potentiated startle (z-score) were analyzed over the entire retrieval session and per burst presentation, respectively. Control 1 (10 seconds CS, predictable CS US timing) To assess the importance of the unpredictability of a stimulus for sustained fear responses, a subset of C57BL/6J animals (n = 20) was subjected to sustained fear training with altered conditions. In this protocol, animals were confronted to four 10 khz tone presentations at 75 db with constant (predictable) duration of 10 seconds. Each tone was followed by a footshock (US, 0.4 ma, 1 seconds). Inter-stimulus intervals were pseudo-randomized (15, 20 and 19 seconds) and presented in a different order between the first and the second training session 6 h later. Trained animals were separated into two groups for retrieval session (as performed in the main experimental group shown above), with one group comprising animals that were confronted to predictable CS paired with burst presentations (predictable, CS + burst; n = 10) and another group serving as CS-only control group (predictable, CS-only group; n = 10) (see schematic drawing in Fig. 5a). 284 Genes, Brain and Behavior (2015) 14: 281 291

Phasic and sustained fear in mice pseudo-randomized intervals (29, 9, 19 and 14 seconds) followed by four CS presentations (first CS 30 seconds after termination of the last US). Conditioned stimulus of variable length (pseudo-randomized arrangement: 29, 9, 19 and 14 seconds) with a fixed 30 seconds ISI (starting with termination of the previous CS) were presented. This session was repeated 6 h later with different order of pseudo-randomized CS durations. For retrieval session (same as in the main group shown above), animals were divided into 2 groups [unpaired, CS + bursts (n = 10) and CS-only (n = 10) group] (see schematic drawing in Fig. 5d). Figure 4: Freezing and fear-potentiated startle after fear conditioning using CS US pairings at variable duration. (a) Schematic representation of fear conditioning procedure. (b) The fraction of time spent freezing (% of 30 seconds time bins) before, during and after CS in the presence of startle-eliciting stimuli (CS + burst, filled circles) compared to CS presentation in absence of bursts (CS-only, open circles). Note that CS presentation in combination with startle stimuli induces strong freezing and leads to prolonged and sustained freezing throughout CS presentation (solid horizontal line indicates significant values identified by Wilcoxon matched pairs signed-rank test). Exclusive CS presentation induces initial or phasic freezing responses with rapid decrease of freezing behavior (dotted horizontal line indicates significant values identified by Wilcoxon matched pairs signed-rank test). (c) Fear-potentiated startle (z-scored data with mean startle amplitude during adaptation serving as reference) before, during and after CS presentation indicates significantly increased startle amplitudes during presentation of the CS in presence of startle stimuli, thereby matching the temporal progression of freezing responses during CS presentation. Furthermore, paired t-test analysis of z-score data with pre-cs period as reference shows that startle responses are significantly fear-potentiated throughout tone (solid horizontal line). Values are mean + SEM. CS + burst: n = 18, CS-only: n = 12. Control 2 (CS US unpaired training) To assess unconditioned effects of auditory background tone (10 khz) presentations on startle responses to noise burst presentations per se, an additional group of C57BL/6J mice (n = 20) underwent training with an unpaired CS US presentation. In this protocol, animals received four 0.4 ma scrambled footshocks (1 seconds duration) in Statistics Analysis of variance (ANOVA) with repeated measurements was used to analyze freezing behavior between groups followed by Bonferroni post hoc test for multiple comparisons. Freezing periods of 30 seconds bins served as within-subject factor for repeated measures during retrieval. Freezing behavior of individual groups was interpreted utilizing two-tailed Wilcoxon signed-ranks test for matched pairs with pre-cs time period as reference, because freezing during pre-cs period was only barely or not detectable. Startle responses during retrieval were evaluated by z-score analysis. For each animal, the startle amplitude of each burst (36 in total) during retrieval was subtracted by the mean of all startle amplitudes during adaptation and divided by the standard deviation of the adaptations mean. Analysis of variance with repeated measurements was chosen to analyze startle behavior between groups followed by Bonferroni post hoc test for multiple comparisons based on z-score data. In addition, to further detect significant changes in startle amplitudes, a paired t-test of z-values was performed with pre-cs time period as reference. All values are expressed as mean ± standard error of mean (SEM). Results The paradigm used for distinction between phasic and sustained fear components consisted of the following major steps (Fig. 1; for details see material and methods): In a pre-test session, startle threshold and intensity of auditory white noise burst stimuli needed to evoke 50% of the maximum startle amplitude were determined in an individual animal. In the adaptation session (day 1), animals were adapted to the test apparatus in context A and exposed to the pre-determined startle-provoking white noise bursts. On day 2, auditory Pavlovian fear conditioning was performed in a novel context (B), with pseudo-randomized CS (tone) US (footshock) presentations at various durations. On day 3, fear retrieval was tested by presentation of the CS at 6 min duration, with or without repetitive white noise burst presentation in different groups of animals. Two separate groups of animals were used as controls: one group (control 1) with predictable timing of the unconditioned stimuli (US), in which the influence of predictability of the CS on fear expression was tested; a second group (control 2) with explicitly unpaired shock-tone (US CS) presentations to investigate Genes, Brain and Behavior (2015) 14: 281 291 285

Daldrup et al. Figure 5: Freezing and fear-potentiated startle after fear conditioning using CS US pairing at constant duration (a-c; control 1) and explicitly unpaired CS US (d-f; control 2). (a) Schematic representation of training procedure, using CS US pairing at constant duration. (b) Fraction of time spent freezing (% of 30 seconds time bins) before, during and after CS presentation. Both groups (CS + burst, filled circles, CS-only, open circles) show increased freezing after CS onset with fast decay during CS presentation (solid horizontal line indicates significant values identified by Wilcoxon matched pairs signed-ranks test). (c) Prolonged startle responses during CS + burst presentation (z-scored data with mean startle amplitude during adaptation as reference; solid horizontal line indicates significant values identified by paired t-test). (d) Training procedure with explicitly unpaired CS US presentation. (e) Fraction of time spent freezing (% of 30 seconds time bins) before, during and after CS presentation. Note only minor and transient freezing in the CS+ burst group (filled circles) after CS onset (solid horizontal line indicates significant values identified by Wilcoxon matched pairs signed-ranks test), while no significant freezing is detected in the CS-only group (open circles). (f) Startle responses (z-scored data with mean startle amplitude during adaptation as reference) after unpaired training show only minor and transient fear potentiation at early CS presentation (solid horizontal line indicates significant values identified by paired t-test). Values are mean + SEM. Animals in each group n = 10. unconditioned effects of the stimuli and a putative influence of the auditory CS background environment. To determine the stimulus threshold, which evoked 50% of the mean maximum startle response (pre-test), the following stimulus intensity-dependent mean startle amplitudes were identified: 55 db: 76.06 ± 1.25; 65 db: 85.71 ± 1.52; 75 db: 113.9 ± 2.91; 85 db: 206.2 ± 9.23; 95 db: 393.0 ± 22.63; 100 db: 420.5± 10.44 AU. This pre-test revealed a mean burst intensity of 88.24 ± 0.56 db sound pressure level (SPL) (range 85 95 db) to evoke 50% of maximal startle amplitude (n = 70 mice). During adaptation, mice displayed a mean startle amplitude of 276.59 ± 15.76 (arbitrary units) to 36 delivered, individually calibrated white noise bursts. Comparison of mean startle amplitudes of different groups (main group, n = 30: 317.09 ± 28.23; control 1, n = 20: 238.95 ± 25.54; control 2, n = 20: 253.48 ± 21.21) revealed no significant difference (ANOVA; F 2,67 = 2.658, P = 0.0775), indicating that during adaptation startle amplitudes were similar across animals. In a next set of experiments, the influence of white noise bursts on conditioned fear was tested to investigate freezing and fear-potentiated startle of C57BL/6J mice (n = 18) exposed to the CS in the presence of 36 individually calibrated white noise burst presentations (main group, CS + burst) compared to control animals exposed to a 6 min CS presentation in absence of bursts (main group, CS-only, n = 12). As illustrated in Fig. 4, animals of the CS + burst group displayed a prolonged and sustained freezing response throughout the entire CS presentation compared to animals of the CS-only group, which only showed an initial transient (phasic-like) high freezing response to the CS presentation, that rapidly decreased within 2 min after CS onset. 286 Genes, Brain and Behavior (2015) 14: 281 291

Phasic and sustained fear in mice Two-way ANOVA with repeated measurements [CS + burst and CS-only group as between-subject factor, and 36 time bins (30 seconds each) as within-subject factor] revealed a significant effect of within-subject (F 35,980 = 107.9, P < 0.0001), between-subject subject (F 1,28 = 93.58, P < 0.0001) and interaction between both factors subject (F 35,980 = 23.29, P < 0.0001) throughout fear memory retrieval. Bonferroni post hoc test for multiple comparisons indicated significantly higher freezing during CS presentation of CS + burst group compared to CS-only group (time bins 13 24, P < 0.0001; time bin 25, P < 0.001). Furthermore, no freezing differences were observed in pre- and post-cs presentation periods (Fig. 4b). Additionally, comparison of freezing following CS onset against baseline (pre-cs period) revealed highly significant and sustained freezing response throughout tone presentation for the CS + burst group (Wilcoxon matched pairs signed-ranks test: time bins 13 24, P < 0.0001; time bin 25, P < 0.001), indicating a sustained state of fear (Fig. 4b). Conditioned stimulus-only group showed significantly elevated, but short-lasting freezing upon CS presentation, indicating a rather phasic state of fear (Wilcoxon matched pairs signed-rank test: time bins 13 14, P < 0.001; time bin 15, P < 0.01). Taken together, these results suggest a major influence of burst presentations on the temporal fear profile, revealing a putative transition phase from phasic to sustained fear within 2 min after CS onset. Z-score analysis of startle responses during retrieval revealed significantly increased fear-potentiated startle during CS presentation (z-score > 1.96 and paired t-test: time bin 13, P < 0.0001; time bins 14 15, P < 0.01; time bin 16, P < 0.001; time bin 17, P < 0.01; time bin 18, P < 0.001; time bins 19 20, P < 0.01; time bins 21 22, P < 0.05; time bins 23 24, P < 0.01) of the CS + burst group (Fig. 4c). No startle amplitude differences were observed in pre- and post-cs presentation periods. To assess the influence of predictability vs. unpredictability of CS US timing on fear expression, we evaluated the effect of training to short-lasting CS (10 seconds) with predictable timing of the US (control 1, Fig. 5a c). After pre-testing and adaptation, animals were conditioned to 10 seconds tone presentations each followed by footshock (0.4 ma, 1 second duration). Fear memory retrieval during CS presentation was tested in two subgroups, with (control 1, predictable, CS + burst, n = 10) and without coinciding burst presentation (control 1, predictable, CS-only, n = 10). Following tone onset, animals of both groups displayed strong initial, but transient freezing, dissipating rapidly within 2 min (Fig. 5b). Two-way ANOVA with repeated measurements (CS + burst and CS-only group as between-subject factor, and 36 time periods of 30 seconds as within-subject factor) discovered a significant effect of within-subject (F 35,630 = 157.6, P < 0.0001), between-subject (F 1,18 = 9.462, P < 0.01) and interaction between both factors (F 35,630 = 3.56, P < 0.0001) during retrieval. Bonferroni post hoc test for multiple comparisons indicated a significantly higher freezing during CS presentation of the CS + burst group compared to the CS-only group (time bins 14 16, P < 0.0001; time bin 17, P < 0.01, time bin 18, P < 0.05; Fig. 5b). Furthermore, a Wilcoxon signed-rank test for matched pairs revealed that CS + burst animals displayed significantly higher freezing responses during the first period of tone presentation (time bins 13 18, P < 0.01; time bin 20, P < 0.05) if compared to pre-cs freezing behavior, CS-only group showed significantly elevated freezing from time bin 13 to 16(P < 0.01, Fig. 5b). Also, z-score analysis of startle responses during fear retrieval (adaptation as reference) of CS + burst animals revealed significantly increased fear-potentiated startle initially after CS onset (z-score > 1.96 and paired t-test: time bin 13 and 15, P < 0.05; time bin 16, P < 0.01; time bins 17 19, P < 0.05; time bin 20, P < 0.01; time bins 22 23, P < 0.05). Differences in startle amplitude were not observed during pre- and post-cs presentation periods (Fig. 5c). Another group of animals (n = 20) underwent pre-testing, adaptation and unpaired training to unravel potential unconditioned effects of the auditory CS on fear behavior (control 2, Fig. 5d f). Animals were subdivided into CS + burst (n = 10) and CS-only group (n = 10) and were tested for fear memory retrieval as described above. Animals of both groups displayed initial, relatively low freezing responses (within first 2 min) during CS presentation (Fig. 5e). Two-way ANOVA with repeated measurements (CS + burst and CS-only group as between-subject factor, and 36 time periods of 30 seconds as within-subject factor) revealed a significant effect of within-subject (F 35,630 = 5.929, P < 0.0001), between-subject (F 1,18 = 8.282, P < 0.01), but no interaction between both factors (F 35,630 = 1.123, P = 0.2903) during retrieval. Bonferroni post hoc test for multiple comparisons indicated a significantly higher freezing within 1 min after CS onset of the CS + burst group compared to the CS-only group (time bin 13, P < 0.01; time bin 14, P < 0.05). Wilcoxon matched pairs signed-rank test showed significantly elevated, but only short lasting freezing (time bin 13 and 15, P < 0.001; time bin 14, P < 0.05) for CS + burst animals, while no significant differences were detected for CS-only group. Furthermore, z-score analysis of startle response for CS + burst group revealed weak but significant effects, only in response to the first bursts after CS onset (z-score > 1.96 and paired t-test: time bin 13 and 15, P < 0.01) (Fig. 5f). Analysis of variance with repeated measurements of CS-evoked freezing between all CS + burst groups (fear conditioning to unpredictable and predictable CS US timing and unpaired CS US), with groups as between-subject factor and 36 time periods of 30 seconds as within-subject factor revealed a significant effect of within-subject (F 35,1225 = 131.1, P < 0.0001), between-subject (F 2,35 = 103.2, P < 0.0001) and interaction between both factors (F 70,1225 = 36.95, P < 0.0001). Post hoc analysis revealed that freezing in the main group (unpredictability, Fig. 4b black solid line) was significantly increased in comparison to control 1 (predictability, time bins 15 25, P < 0.0001, Fig. 5b black solid line) and control 2 (unpaired, time bins 13 25, P < 0.0001, Fig. 5f black solid line). Comparison between the two control groups revealed significant freezing in control 1 with a transient time course (time bins 13 16, P < 0.0001; time bin 17, P < 0.001; time bin 18, P < 0.01). Taken together, these results suggest a major influence of training procedure on the temporal fear profile in that conditioning with unpredictable CS US timing (main group) results in sustained, while conditioning with Genes, Brain and Behavior (2015) 14: 281 291 287

Daldrup et al. predictable CS US timing (control 1) results in more phasic fear responses. By contrast, CS presentation after unpaired training (control 2) evokes only minor freezing. Comparison of startle responses of all CS + burst groups in two-way ANOVA with repeated measurements (groups as between-subject factor, and 36 time periods of 30 seconds as within-subject factor) revealed a significant effect of within-subject (F 35,1225 = 13.47, P < 0.0001), no effect of between-subject (F 2,35 = 3.223, P = 0.0519) and a significant interaction between both factors (F 70,1225 = 2.388, P < 0.0001). Post hoc analysis revealed that animals trained to an unpredictable CS US timing (main group, Fig. 4c) showed higher fear-potentiated startle (time bin 13, P < 0.05; time bins 14 15, P < 0.0001; time bin 16, P < 0.001; time bin 17, P < 0.0001; time bin 18, P < 0.01; time bins 19 20, P < 0.05) upon CS presentation when compared to the unpaired training group (control 2, Fig. 5f). Predictable CS US training (control 1, Fig. 5c) significantly increased fear-potentiated startle (time bin 13, P < 0.0001; time bin 14, P < 0.001; time bin 15, P < 0.0001; time bin 17, P < 0.01; time bin 18, P < 0.001; time bin 19, P < 0.01; time bin 20, P < 0.05) in comparison to unpaired training. No startle amplitude differences were observed between main group (unpredictability) and control group 1 (predictability). Taken together, sustainment of fear-potentiated startle occurred upon CS presentation irrespective of predictability or unpredictability of the US during training. Conditioned stimulus presentation after unpaired training (control 2) evoked only minor startle reflexes compared to fear-potentiated responses of the main group and control group 1. Discussion This study describes a dedicated training protocol to assess temporal profiles of conditioned fear in freely behaving C57BL/6J mice. An auditory Pavlovian fear conditioning paradigm with pseudo-randomized CS US presentations at various durations is combined with repetitive brief auditory startle stimuli during fear retrieval to overcome shortcomings of existing methods in several aspects: (1) A setup allowing startle reflex measurements in non-restrained mice was designed and validated, which offers the possibility to describe startle responses in freely moving animals. (2) A behavioral protocol initially developed by Davis et al. to study phasic and sustained fear in rats (Davis et al. 2010) was adopted for application in mice, to pave the way for assessment of phasic/sustained fear transitions in mice and genetically engineered mouse lines. (3) Experiments in dedicated control groups were performed to allow for discrimination between overall influences of auditory background and predictability vs. unpredictability to be able to better define the term sustained fear. (4) Conditioned freezing was measured in parallel to fear-potentiated startle responses, and the results show that sustained components of fear show responsiveness in both behavioral readouts, with sustained freezing reflecting unpredictability of CS US pairings. Pavlovian fear conditioning is considered as an effective paradigm to investigate molecular and neural mechanisms of fear and anxiety in humans and rodents (LeDoux 2003; for review, see Pape & Pare 2010). In this experimental design, the subject associates a CS (e.g. tone) with an aversive unconditioned event (US, e.g. electric footshock). The successful association between CS and US is commonly tested by the presentation of the CS alone, using time spent with freezing and/or startle responses as measures of fear. Freezing is a defensive response after fear conditioning (Fanselow 1980; Fanselow 1994; Fendt & Fanselow 1999; Maren & Fanselow 1996), which has been defined as absence of movement except for respiration. Fear-potentiated startle responses are defined as an elevation of the startle amplitude in presence vs. absence of the CS (Brown et al. 1951; Davis 1986; Davis & Astrachan 1978; Davis et al. 2010; Fendt & Koch 2013). Both responses are commonly used as behavioral readouts to quantify conditioned fear (see Grillon 2002). Fear-potentiated startle is commonly used in rat and human studies (Davis et al. 2010; Grillon & Davis 1997), and together with closely resembling training parameters, constitute a unique approach to translational research on learning and memory processes across species (Falls 2002). In mice, freezing is the predominant measure of fear and only few studies have reported on fear-potentiated startle responses, which is notoriously difficult to study in this species (see for instance, Crawley & Paylor 1997; Fendt & Koch 2013). The apparatus described in this study allows measurement of startle responses in a semi-quantitative manner in freely moving, non-restrained mice. In contrast to most commercially available startle cages, in which animals are restraint or at least restricted in their natural movements, the main advantage of our motion-sensitive platform is that animals are capable of moving freely, like in an open field arena. The measurement of startle responses is based on three equally distributed mechano-electrical transducers at the bottom of the motion-sensitive plate, which in principle are also used in commercially available startle systems (e.g. SR-LAB, San Diego Instruments, San Diego, CA, USA). The integrated signal and the distribution of the piezo elements assure the measurement of minimal movements of the platform irrespective of the animal s position in the arena. As is illustrated in Fig. 3, the range of sensitivity of this device indicates both high-amplitude startle responses and low-amplitude signals, such as during exploration of the animal. This motion-sensitive apparatus thus allows to distinguish periods of locomotor activity and periods of immobility and may be used in addition to semi-automatized devices for inspection of behavior in experimental animals (Meuth et al. 2013). Our device offers the additional advantage that it is easily accessible to electrophysiological and/or optogenetic recording designs both essential and important techniques for future studies aiming at the neuronal substrates and mechanisms of fear in freely behaving mice. The common experience that startle responses are difficult to assess experimentally in mice has been related, among others, to genetic variances, sensorimotor deficits and age-dependent hearing loss (Falls 2002; Fendt & Koch 2013). One important precondition to reliably evoke startle reflexes in mice is to identify inter-individual differences in sensitivity to white noise burst intensities. Here, we used a pre-test procedure suggested by Falls (2002), where 288 Genes, Brain and Behavior (2015) 14: 281 291

Phasic and sustained fear in mice the identified inter-individual differences can be used to individually adjust startle stimulus intensities in order to avoid ceiling or floor effects. In this study, an individually pre-determined stimulus intensity of about 50% from maximum startle response has proven adequate to reliably evoke fear-potentiated startle and to allow assessment of freezing upon presentation of a 75 db 10 khz auditory CS in a retrieval session. It is noteworthy that the inter-individual variance of pre-determined stimulus intensities was low, and inter-individual differences in mean startle amplitudes during adaptation were not observed. Data from individual pre-testing of only a subpopulation of animals may thus provide a reliable mean for application to the overall population under study. This, however, needs to be determined separately for a population of animals in each experimental study. In a classical fear conditioning procedure, short and discrete cues predictably paired with an aversive event form a fear memory which quickly vanishes once the threatening stimulus is no longer present, a transient physiological state usually referred to as a phasic state of fear (Davis et al. 2010). However, situations in patients suffering from anxiety disorders indicate the opposite, a more long-lasting, sustained state of fear, wherefore it is generally assumed that clinical symptoms of several anxiety disorders can be modeled more precisely with paradigms aiming on these sustained, rather than phasic fear states. Walker and Davis initially developed a paradigm in rats, where a training paradigm is described, in which rats were trained with CS (60 Hz-clicker auditory stimuli) of variable duration followed by a footshock (US), imposing unpredictability on the US (Davis et al. 2010). During retrieval, startle inducing bursts were applied, once in the presence of a clicker-tone for 8 min and once in absence of clicker presentation. Results show an abrupt increase of startle amplitudes, followed by a quick decline, indicating the phasic period of fear, followed by a more maintained elevation of startle defined as sustained fear. In a subsequent study, Davis and coworkers used a protocol slightly modified from their initial one and reported on various time courses of fear-potentiated startle responses to introduce the terms phasic fear and sustained fear for description of the rapidly declining and more maintained states of fear-potentiated startle (Miles et al. 2011). To reveal temporal profiles of conditioned fear in mice, we adopted the aforementioned paradigm and included specific modifications to make it applicable to mice. Rather than using a 60 Hz-clicker presentation for 8 min as CS (as used in studies in rats), we delivered a 10 khz sine wave lasting for 6 min as CS. Although presentations of different tone frequencies may also influence behavioral readouts, the 10 khz sine wave represents an established stimulus for fear conditioning in mice (Lesting et al. 2011; Lesting et al. 2013; Seidenbecher et al. 2003). Testing animals 24 h after fear conditioning revealed that phasic and sustained components of fear can be evoked and measured in mice using fear-potentiated startle and freezing behavior in parallel as measures of fear during presentation of a fear-conditioned tone in conjunction with startle-inducing bursts. Conditioned stimulus presentation in combination with startle stimuli readily induced strong freezing and startle responses. Furthermore, data revealed prolonged and sustained elevated behavioral responses throughout CS presentation. The temporal profile of both behavioral readouts, with strong responses immediately after CS onset and more moderate but still elevated response magnitudes during ongoing CS, resembled the time course of conditioned fear which was introduced as phasic and sustained fear by Davis et al. (Davis et al. 2010). Interestingly, CS-only presentation induced only initial or phasic fear responses with rapid decrease in magnitude within the first 2 min after CS onset. Two control groups were tested in an additional series of experiments: Experiments on one group with explicitly unpaired shock-tone (US CS) presentation aimed at revealing unconditioned effects of stimuli and possible influences of the background auditory environment (CS); in a second group with predictable timing of the US, the influence of predictability vs. unpredictability of the CS on fear expression was tested. In the unpaired group, only initial and weak freezing and fear-potentiated startle responses were observed, indicating that unconditioned stimuli or background auditory environment (CS presentation) did not affect behavioral responses evoked by burst presentations in an unconditioned training paradigm. Furthermore, we did not observe startle attenuation during CS presentation, which would be the case if the animals associated the CS with safety (Pollak et al. 2010) or relief (Gerber et al. 2014), and therefore we consider this as a neutral control. In the group with predictable US timing, freezing rapidly declined within 2 min after CS onset, very much resembling the temporal course of the phasic freezing type observed in mice trained with unpredictable CS US pairings but no concomitant startle bursts. Animals of the main group, tested with simultaneous CS and burst presentations, displayed a sustained fear response, allowing us to conclude that US unpredictability during conditioning and simultaneous CS/startle burst-presentations during retrieval are necessary for sustainment of conditioned freezing. One important point is that habituation-like phenomena may contribute to the phasic-sustained fear response profile, and that the startle stimuli specifically serve to dishabituate freezing. Repeated non-reinforced stimulus presentations have indeed been observed to decrease the responsiveness to the CS due to habituation-like processes (Kamprath & Wotjak 2004; McSweeney & Swindell 2002). Similar habituation phenomena might be induced by the long duration CS and thereby contribute to a phasic fear phenotype, while concomitant repetitive burst stimuli interfere with these processes resulting in dishabituation and more sustained fear responses. Results from control experiments using CS with predictable US training argue against this possibility, in that phasic freezing response profiles prevail irrespective of the presence or absence of repetitive burst stimuli. Another important notion is that fear-potentiated startle responses displayed sustained components, irrespective of the training with predictable CS US pairings or US unpredictability. There are some major lines of conclusions. First, freezing and fear-potentiated startle involve different neuronal circuits and neurochemical mechanisms which only partly overlap (reviewed by Fendt & Fanselow 1999; Santos et al. 2005). One possible consequence being that predictable aversive stimuli and startle provoking bursts are differently Genes, Brain and Behavior (2015) 14: 281 291 289

Daldrup et al. processed in the respective networks, resulting in divergent temporal profiles of behavioral output signals phasic components of freezing and sustained-type potentiation of startle responses. Second, increases in amplitude and duration of startle responses may result from an increase in sensitivity (hypersensitivity) by the conditioning procedure irrespective of predictability or unpredictability of CS US pairing (Davis 1989; Richardson 2000). The small amplitudes of startle responses in the unpaired control group support this conclusion. Third, the sustained component of freezing cannot be considered a mere consequence of startle-stimuli present during testing of the CS, or the long duration of CS presentation. Rather, sustained components of freezing require unpredictable CS US conditioning paradigms, similar to previous observations made in rat and human studies discussed above (Davis et al. 2010; Grillon & Davis 1997). The sustainment of conditioned freezing in mice thus seems to reflect an effect of learning or retrieval of fear, rather than a performance effect where the presence of sensitizing sensory stimuli may sustain conditioned fear or prevent habituation-like processes. As a corollary, the temporal response profile of conditioned freezing upon unpredictable CS US training is thus an important behavioral output variable when it comes to the analysis of the neurobiological mechanisms or pharmacological features underlying phasic and sustained components of learned fear in mice. Finally, studies in rats have indicated that startle responses are suppressed by intense freezing, as if the animals are paralyzed in fear (Davis & Astrachan 1978), while others found startle-responses to be facilitated by freezing (Leaton & Borszcz 1985; Plappert et al. 1993), suggesting species-specific differences or influences of the specific experimental paradigms. 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